Folypoly-gamma-glutamate synthetase will be purified from a variety of bacterial and mammalian sources, and its substrate specificity and kinetic properties examined to determine whether they are responsible for the different types of pteroylopolyglutamates found in different cell lines. Pteroylpolyglutamate substrates and potential analog inhibitors of the synthetase will be synthesized by a solid phase procedure. The effects of products of one-carbon metabolism, such as methionine, thymidine, and adenosine, on the folate requirements, the glutamate chain lengths of endogenous folates, and on folypoly-gamma-glutamate synthetase levels will be examined in bacteria and a variety of tumorigenic and nontumorigenic mammalian cells. These studies will investigate whether one-carbon metabolism is controlled by varying the glutamate chain length of endogenous pteroylpolyglutamates and whether differences exist in folate requirements and requirements for products of one-carbon metabolism between normal and malignant cells. Inhibitors of folypoly-gamma-glutamate synthetase will be tested for their ability to cause cellular folate depletion by preventing the formation of pteroylpolyglutamates, the forms of vitamin that are selectively retained by tissues.